The covalent joining of nucleic acid probes by ligase enzymes is one of the most useful tools available to molecular biologists. When two probes are annealed to a template nucleic acid where the two probes are adjacent and without intervening gaps, a phosphodiester bond can be formed by a ligase enzyme (Whiteley, 1989). The ligation bond is formed between a 5' terminus of one probe and the 3' terminus of the other probe.
The events of annealing and ligation each require a high level of fidelity, i.e. complementarity, between the sequences of the ligating probes and the template nucleic acid. Both events are inefficient when base-pairing mismatches occur. Generally, DNA ligase can join two adjacent probes only when they perfectly complement a denatured template nucleic acid, such as a PCR product (Landegren, 1988; Nickerson, 1990). Even a single nucleotide mismatch at, or near, the ligation site of the probes will prevent ligation of the annealed probes.
Oligonucleotide ligation assays detect the presence of specific sequences in target DNA sample. For example, allelic discrimination assays rely on probes representing the complementary sequences of the allelic forms to the target. Ligation to a common, second target-complementary probe indicates the presence of the polymorphic site (Whiteley, 1989; Landegren, 1988). Absence of ligation indicates the lack of the polymorphic site. Ligation can be detected through detectable labels on the allelic probe and electrophoretic separation of the ligation products (Grossman, 1994).
It is desirable to provide optimized probes and methods of annealing and ligation. Such methods would improve assays and tests that benefit from greater precision and accuracy.